Background: Visceral Leishmaniasis is endemic to Brazil, where it is caused by Leishmania infantum (syn. Leishmania chagasi). Following parasite inoculation, individuals may experience asymptomatic infection, raising the possibility of parasite transmission through the transfusion of contaminated blood products. In the present work, we evaluated the prevalence of asymptomatic Leishmania infection among blood donors in Salvador, northeastern Brazil. Methods: Peripheral blood was collected from 700 blood donors attending the Blood Bank of Bahia (HEMOBA/SESAB), from January to September 2010. We evaluated anti-Leishmania serology by ELISA, employing Soluble Leishmania Antigen (sensitivity 90% and specificity 95%). The presence of parasite DNA was determined by qPCR, targeting a single copy gene (G6PD), and by end-point PCR, targeting multiple targets, namely a segment located in the Leishmania rRNA locus (ITS) and the conserved region of kinetoplastid DNA (kDNA) minicircles. Results: The blood-donor population was comprised of 74.5% of males with a mean age of 34 years. Anti-Leishmania serology by ELISA was positive in 5.4% (38/700) individuals. One individual was also positive for Chagas’ disease and another tested positive for Syphilis. Employing qPCR, parasite DNA was not found in any of 38 seropositive samples. However, by ITS PCR, 8/38 (21%) samples were positive and this positivity increased to 26/38 (68%) when we targeted kDNA amplification. Agreement between both techniques (ITS and kDNA PCR) was fair (kappa = 0.219). Conclusions: These results indicate that asymptomatic infection is present among the blood donor population of Salvador, a finding that warrants a broader discussion regarding the need to implement specific screening strategies.